Pig-derived tendon decellularized material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tissue repair materials, and particularly discloses a porcine tendon decellularized material as well as a preparation method and application thereof. The preparation method of the pig tendon decellularized material disclosed by the invention specifically comprises the following steps: sequentially carrying out repeated freezing and thawing treatment, treatment with a TritonX-100 solution with the concentration of 0.5 v / v% to 1.5 v / v%, treatment with a SDS solution with the concentration of 0.5 wt% to 1.5 wt%, treatment with a TritonX-100 solution with the concentration of 1.5 v / v% to 3 v / v%, treatment with a sodium deoxycholate solution with the concentration of 2 wt% to 3.5 wt%, treatment with a DNaseI enzyme solution with the concentration of 50 U / mL to 150 U / mL, treatment with a 100% isopropanol solution, and freeze-drying, so as to obtain the pig tendon decellularized material. Through the preparation method, the pig-derived tendon decellularized material prepared by the application has relatively low content of DNA and has excellent mechanical properties.

Description

Technical Field

[0001] The present application relates to the technical field of tissue repair materials, and in particular to a porcine tendon decellularized material and a preparation method and application thereof. Background Art

[0002] Anterior cruciate ligament rupture is one of the most common sports injuries, and its incidence is also increasing. Arthroscopic reconstruction of the anterior cruciate ligament is currently the most commonly used surgical method. The sources of ligaments required for reconstruction are mainly autologous tendons, allogeneic tendons and artificial ligaments. In most cases, autologous tendon grafts do not worry about immune rejection and disease transmission, but the operation time is long and the trauma is greater. The ligaments after allogeneic tendon reconstruction are strong and recover quickly after surgery, but there is a risk of rejection. Artificial ligaments are expected to avoid the loss of autologous structure and the spread of disease, but they are expensive and have weak bone integration ability. In the long term, a very small number of patients will be at risk of rupture and loosening. Therefore, it is crucial to develop a ligament that has matching mechanical strength, good therapeutic effect and low price.

[0003] Recent studies have shown that porcine tendons have mechanical strengths that match those of human ligaments, and that porcine ligaments are widely available, low cost, and have good repair effects, and therefore have great potential for application in the repair of anterior cruciate ligament ruptures. However, related research is only in its early stages, and the evidence is not sufficient, and the process is not mature. Therefore, it is urgent to further explore the role of this material in the repair of anterior cruciate ligament ruptures. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a porcine tendon decellularized material and a preparation method and application thereof.

[0005] In the first aspect, the present application provides a method for preparing a decellularized material of porcine tendon, which specifically comprises the following steps: subjecting the porcine tendon sample to repeated freeze-thaw treatment, 0.5v / v%-1.5v / v% TritonX-100 solution treatment, 0.5wt%-1.5wt% SDS solution treatment, 1.5v / v%-3v / v% Triton X-100 solution treatment, 2wt%-3.5wt% sodium deoxycholate solution treatment, 50-150U / mL DNaseI enzyme solution treatment, 100% isopropanol solution treatment, and freeze-drying to obtain the obtained material.

[0006] The present application prepares a porcine tendon decellularized material by subjecting a porcine tendon sample to repeated freeze-thaw treatment, treatment with a low concentration of TritonX-100 solution, treatment with an SDS solution, treatment with a high concentration of TritonX-100 solution, treatment with a sodium deoxycholate solution, treatment with a 50-150U / mL DNaseI enzyme solution, treatment with a 100% isopropanol solution, and freeze-drying. The finished material has a low DNA content and excellent mechanical properties.

[0007] Among them, Triton X-100 is a detergent that can effectively remove cell membrane lipids and further remove cell contents. This concentration range is considered to be able to effectively remove cell membranes without causing significant damage to the matrix; SDS solution is a strong detergent that helps to completely remove cell membranes and intracellular proteins. This concentration is sufficient to ensure the thoroughness of decellularization while avoiding excessive damage to the structure of the matrix; using a higher concentration of Triton X-100 can further remove unremoved cellular components and avoid excessive damage to the structure of porcine tendon samples; sodium deoxycholate solution treatment helps to remove membrane lipids and enhance the decellularization effect. This concentration can balance the delipidation effect with the effect on the matrix; DNaseI enzyme solution treatment is used to degrade DNA in cells to prevent immune reactions in subsequent treatments. The concentration of the enzyme is selected based on its ability to effectively degrade DNA within a specific time to ensure efficiency and safety; 100% isopropanol solution treatment is used to remove moisture and maintain the structure of the decellularized material.

[0008] This application designs and compares different decellularization methods to select the decellularization method with the best comprehensive performance; ultimately stabilizes the process and improves the treatment effect, providing patients with anterior cruciate ligament tears with another low-cost artificial ligament with excellent treatment effect; the above-mentioned technical scheme completely decellularizes the cells on the porcine tendon sample tissue, and the conditions are mild, fast and stable; the resulting decellularized material can simulate the layered structure of natural tendon composite muscle, better retains the extracellular matrix components, and also has the advantages of extremely low immunogenicity and strong mechanical properties, and can be used as artificial ligament repair materials.

[0009] Preferably, the specific steps of the repeated freeze-thaw treatment are: immersing the sample in liquid nitrogen for 3-10 minutes, and then thawing it in pure water at 37° C. for 5-15 minutes; repeating the above steps 3-6 times in total.

[0010] Preferably, the specific steps of the 0.5 v / v%-1.5 v / v% Triton X-100 solution treatment are: immersing the sample in the 0.5 v / v%-1.5 v / v% Triton X-100 solution, stirring on a shaker for 6-12 hours, and cleaning the sample by ultrasound.

[0011] Preferably, the specific steps of the 0.5wt%-1.5wt% SDS solution treatment are: washing the sample with 0.5wt%-1.5wt% SDS solution, stirring for 6-12 hours, and washing the sample by ultrasound.

[0012] In some specific embodiments, the concentration of the SDS solution may be 0.5wt%-1.0wt%, 1.0wt%-1.5wt%.

[0013] In a specific embodiment, the concentration of the SDS solution may also be 0.5wt%, 1.0wt%, or 1.5wt%.

[0014] Preferably, the specific steps of the 1.5 v / v%-3 v / v% Triton X-100 solution treatment are: placing the sample in a 1.5 v / v%-3 v / v% Triton X-100 solution, stirring for 8-12 hours, and cleaning the sample by ultrasound.

[0015] In some specific embodiments, the concentration of the TritonX-100 solution can be 1.5 v / v%, 2.0 v / v%, 2.5 v / v%, 2.8 v / v%, or 3 v / v%.

[0016] In a specific embodiment, the concentration of the TritonX-100 solution can also be 1.5-2.0v / v%, 1.5-2.5v / v%, 1.5-2.8v / v%, 2.0-2.5v / v%, 2.0-2.8v / v%, 2.0-3v / v%, 2.5-2.8v / v%, 2.5-3v / v%, 2.8-3v / v%.

[0017] Through experimental analysis, it can be known that the present application uses the above-mentioned concentration of TritonX-100 solution to treat the sample to obtain a porcine tendon decellularized material with better performance.

[0018] Preferably, the specific steps of treating with 2wt%-3.5wt% sodium deoxycholate solution are: treating and soaking the sample with 2wt%-3.5wt% sodium deoxycholate solution, stirring for 6-24h, and cleaning the sample by ultrasound.

[0019] In some specific embodiments, the concentration of the sodium deoxycholate solution can be 2-2.5wt%, 2-2.8wt%, 2-3.1wt%, 2.5-2.8wt%, 2.5-3.1wt%, 2.5-3.5wt%, 2.8-3.1wt%, 2.8-3.5wt%, 3.1-3.5wt%; the stirring time can be 6-10h, 6-16h, 6-20h, 10-16h, 10-20h, 10-24h, 16-20h, 16-24h, 20-24h.

[0020] In a specific embodiment, the concentration of the sodium deoxycholate solution can also be 2wt%, 2.5wt%, 2.8wt%, 3.1wt%, 3.5wt%; the stirring time can also be 6h, 10h, 16h, 20h, 24h.

[0021] Through experimental analysis, it can be known that the present application uses the above-mentioned concentration of sodium deoxycholate solution to treat the sample for a corresponding period of time to obtain a porcine tendon decellularized material with better performance.

[0022] Preferably, the specific steps of treating with 50-150 U / mL DNase I enzyme solution are: treating the sample with 50 to 150 U / mL DNase I enzyme solution for 6-14 hours to remove residual debris, and cleaning the sample by ultrasound.

[0023] In some specific embodiments, the concentration of the DNaseI enzyme solution can be 50-75U / mL, 50-100U / mL, 50-125U / mL, 75-100U / mL, 75-125U / mL, 75-150U / mL, 100-125U / mL, 100-150U / mL, or 125-150U / mL.

[0024] In a specific embodiment, the concentration of the DNaseI enzyme solution can also be 50 U / mL, 75 U / mL, 100 U / mL, 125 U / mL, or 150 U / mL.

[0025] Preferably, the specific steps of the 100% isopropanol solution treatment are: immersing the sample in a 100% isopropanol solution, degreasing for 3-6 hours at 35° C.-40° C. and 200-400 rpm, and ultrasonically washing with deionized water.

[0026] In a second aspect, the present application provides a porcine tendon decellularized material prepared using the above-mentioned preparation method.

[0027] In a third aspect, the present application provides the use of the above-mentioned porcine tendon decellularized material in artificial ligament repair materials.

[0028] In summary, the technical solution of this application has the following effects: The present application designs and compares different decellularization methods to select the decellularization method with the best comprehensive performance; finally, the porcine tendon decellularized material is prepared by subjecting the porcine tendon samples to repeated freeze-thaw treatment, low concentration TritonX-100 solution treatment, SDS solution treatment, high concentration TritonX-100 solution treatment, sodium deoxycholate solution treatment, 50-150U / mL DNaseI enzyme solution treatment, 100% isopropanol solution treatment, and freeze-drying. The finished material has a low DNA content and excellent mechanical properties.

[0029] The above technical scheme completely decellularizes the cells on the porcine tendon sample tissue, and the conditions are mild, rapid and stable; the obtained decellularized material can simulate the layered structure of natural tendon composite muscle, better retain the extracellular matrix components, and also has extremely low immunogenicity and strong mechanical properties, etc., and can be used for artificial ligament repair materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the HE staining result of the porcine tendon decellularized material prepared in Example 1. DETAILED DESCRIPTION

[0031] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application. Example

[0032] Examples 1-9 Examples 1-9 respectively provide a porcine tendon decellularized material and a preparation method thereof.

[0033] The difference between the above embodiments is that the process parameters of each step in the method for preparing the porcine tendon decellularized material are specifically shown in Table 1.

[0034] The method for preparing the acellular artificial ligament in the above embodiment specifically comprises the following steps: Repeated freeze-thaw treatment: Take the pig tendon composite muscle material, cut the tendon into samples of 2.0×2.0×0.2 cm in size according to the fiber direction, immerse the sample in liquid nitrogen for 5 minutes, and then thaw it in pure water at 37°C for 10 minutes; repeat the above steps for a total of 5 times to destroy the cell membrane of the sample.

[0035] Triton X-100 treatment: The samples were immersed in 0.5 v / v%-1.5 v / v% Triton X-100 solution, stirred on a shaker for 6-12 h, and cleaned by ultrasound.

[0036] Sodium dodecyl sulfate (SDS) treatment: The sample was washed with 0.5 wt%-1.5 wt% SDS solution, stirred for 6-12 h, and cleaned by ultrasound.

[0037] Triton X-100 treatment: The sample was placed in a 1.5 v / v%-3 v / v% Triton X-100 solution, stirred for 8-12 h, and cleaned by ultrasound.

[0038] Sodium deoxycholate treatment: The sample was soaked in a 2.0 wt% to 3.5 wt% sodium deoxycholate solution, stirred for 6 to 24 hours, and cleaned by ultrasound.

[0039] DNase I treatment: Treat the samples with 50 to 150 U / mL DNase I solution for 6-14 h to remove residual debris and clean the samples by ultrasound.

[0040] Isopropanol solution treatment: The sample was immersed in 100% isopropanol solution, degreased at 35°C-40°C and 200-400 rpm for 3-6 h, and then ultrasonically washed with deionized water.

[0041] Freeze drying: Freeze the patch sample at -80℃ for 12-24h and then freeze dry it in a vacuum freeze dryer for 48-72h. Storage: Store samples at -10°C to -30°C until use.

[0042] Table 1 Specific process parameters of each step in Examples 1-9 Comparative Example Comparative Examples 1-7 Comparative Examples 1-7 respectively provide a porcine tendon decellularized material and a preparation method thereof.

[0043] The difference between the comparative example and Example 1 is that the process parameters of each step in the method for preparing the porcine tendon decellularized material are specifically shown in Table 2.

[0044] Table 2 Specific process parameters of each step in Comparative Examples 1-7 The remaining process parameters in the comparative example and embodiment 1 are the same.

[0045] Performance testing (1) HE staining results: The results are as follows Figure 1As shown; the results show that the porcine tendon decellularized material prepared by the technical solution of the present application has no residual cells after HE staining and the collagen structure is well preserved.

[0046] (2) DNA content: The test shall be conducted in accordance with the method of YY / T 1876-2023 “Determination of DNA Residue in Animal-Derived Biomaterials for Tissue Engineering Medical Products: Fluorescence Staining Method”.

[0047] (3) Mechanical strength: tested in accordance with YY / T 0965-2014.

[0048] Test results: as shown in Table 3.

[0049] Table 3 Performance test results of porcine tendon decellularized materials in Examples and Comparative Examples Combined with the test results in Table 3, it can be seen that the present application prepares porcine tendon decellularized material by subjecting porcine tendon samples to repeated freeze-thaw treatment, low concentration TritonX-100 solution treatment, SDS solution treatment, high concentration TritonX-100 solution treatment, sodium deoxycholate solution treatment, 50-150U / mL DNaseI enzyme solution treatment, 100% isopropanol solution treatment, and freeze-drying. The finished material has a low DNA content and excellent mechanical properties.

[0050] In Comparative Example 1, no second TritonX-100 solution treatment was performed after the SDS solution treatment, in Comparative Example 2, no sodium deoxycholate solution treatment was performed, and in Comparative Example 3, no second TritonX-100 solution treatment and sodium deoxycholate solution treatment were performed. The performance of the prepared porcine tendon decellularized material was poor.

[0051] By comparing the test results of Examples 1-3 with those of Comparative Examples 4-5, the present application controls the concentration of the SDS solution during the SDS solution treatment to 0.5wt%-1.5wt%, and controls the concentration of the TritonX-100 solution during the TritonX-100 solution treatment to 1.5v / v%-3v / v%, thereby effectively improving the performance of the porcine tendon decellularized material.

[0052] By comparing the test results of Examples 2, 4-9 with Comparative Examples 6-7, the present application controls the concentration of the sodium deoxycholate solution during the sodium deoxycholate solution treatment to 2wt%-3.5wt%, and controls the concentration of the DNaseI enzyme solution during the DNaseI enzyme solution treatment to 50-150U / mL, which effectively improves the performance of the porcine tendon decellularized material. Furthermore, the present application controls the concentration of the sodium deoxycholate solution to 2.5wt%-3.1wt%, which can further improve the performance of the porcine tendon decellularized material.

[0053] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a decellularized material of porcine tendon, characterized in that: Specifically, the method comprises the following steps: subjecting the porcine tendon sample to repeated freeze-thaw treatment, 0.5v / v%-1.5v / v% TritonX-100 solution treatment, 0.5wt%-1.5wt% SDS solution treatment, 1.5v / v%-3v / v% TritonX-100 solution treatment, 2wt%-3.5wt% sodium deoxycholate solution treatment, 50-150U / mL DNaseI enzyme solution treatment, 100% isopropanol solution treatment, and freeze-drying.

2. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of the repeated freeze-thaw treatment are: immersing the sample in liquid nitrogen for 3-10 minutes, and then thawing it in pure water at 37° C. for 5-15 minutes; repeating the above steps 3-6 times in total.

3. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of the 0.5 v / v%-1.5 v / v% TritonX-100 solution treatment are: immersing the sample in the 0.5 v / v%-1.5 v / v% TritonX-100 solution, stirring on a shaker for 6-12 hours, and cleaning the sample by ultrasound.

4. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of the 0.5wt%-1.5wt% SDS solution treatment are: washing the sample with the 0.5wt%-1.5wt% SDS solution, stirring for 6-12 hours, and washing the sample by ultrasound.

5. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of the 1.5 v / v%-3 v / v% Triton X-100 solution treatment are: placing the sample in a 1.5 v / v%-3 v / v% Triton X-100 solution, stirring for 8-12 hours, and cleaning the sample by ultrasound.

6. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of treating with the 2wt%-3.5wt% sodium deoxycholate solution are: treating and soaking the sample with the 2wt%-3.5wt% sodium deoxycholate solution, stirring for 6-24 hours, and cleaning the sample by ultrasound.

7. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of the 50-150 U / mL DNase I enzyme solution treatment are: treating the sample with 50 to 150 U / mL DNase I enzyme solution for 6-14 hours to remove residual debris, and cleaning the sample by ultrasonication.

8. The method for preparing the porcine tendon decellularized material according to claim 1, characterized in that: The specific steps of the 100% isopropanol solution treatment are: immersing the sample in the 100% isopropanol solution, degreasing for 3-6 hours at 35° C.-40° C. and 200-400 rpm, and ultrasonically washing with deionized water.

9. A porcine tendon decellularized material, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the porcine tendon decellularized material according to claim 9 in artificial ligament repair materials.

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